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Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function 1,2 . Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (Ca V s) 3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming Ca V 1 or Ca V 2 Ca V α 1 (ref. 3 ), and the auxiliary Ca V β 5 and Ca V α 2 δ subunits 6,7 . Here we present cryo-electron microscopy structures of human brain and cardiac Ca V 1.2 bound with Ca V β 3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC) 8,9 -and of the assembled Ca V 1.2-Ca V β 3 -Ca V α 2 δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the Ca V α 2 δ-interaction site. The structures identify the Ca V α 2 δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs 6 , show that EMC and Ca V α 2 δ interactions with the channel are mutually exclusive, and indicate that EMC-to-Ca V α 2 δ hand-off involves a divalent ion-dependent step and Ca V 1.2 element ordering. Disruption of the EMC-Ca V complex compromises Ca V function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a Ca V assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Prokaryotic Na V channels are tetramers and eukaryotic Na V channels consist of a single subunit containing four domains. Each monomer/domain contains six transmembrane segments (S1-S6), S1-S4 being the voltage-sensor domain and S5-S6 the pore domain. A crystal structure of Na V Ms, a prokaryotic Na V channel, suggests that the S4-S5 linker (S4-S5 L ) interacts with the C-terminus of S6 (S6 T ) to stabilize the gate in the open state. However, in several voltage-gated potassium channels, using specific S4-S5 L -mimicking peptides, we previously demonstrated that S4-S5 L /S6 T interaction stabilizes the gate in the closed state. Here, we used the same strategy on another prokaryotic Na V channel, Na V Sp1, to test whether equivalent peptides stabilize the channel in the open or closed state. A Na V Sp1-specific S4-S5 L peptide, containing the residues supposed to interact with S6 T according to the Na V Ms structure, induced both an increase in Na V Sp1 current density and a negative shift in the activation curve, consistent with S4-S5 L stabilizing the open state. Using this approach on a human Na V channel, hNa V 1.4, and testing 12 hNa V 1.4 S4-S5 L peptides, we identified four activating S4-S5 L peptides. These results suggest that, in eukaryotic Na V channels, the S4-S5 L of DI, DII and DIII domains allosterically modulate the activation gate and stabilize its open state.